Regulation of gene transcription1. Three-stage regulation of the amphibolic gal regulaon.The gal operon of Escherichia coli is negatively regulated by the Gal repressosome, a higher order nucleoprotein complex containing a DNA loop that encompasses two gal promoters. In the repressosome structure, Gal repressor (GalR) dimers are bound to the two operator sites, flanking the promoter region, thus generating a DNA loop. The DNA loop is stabilized by binding of the architectural HU protein to the apex of the loop, and negative supercoiling. The gal promoters are also regulated in opposite directions by GalR without DNA looping. The repressosome-mediated as well as looping-independent transcription regulation of the two promoters is lifted in the presence of the inducer D-galactose. We tested the effect of D-galactose on various DNA-protein and protein-protein interactions of different regulatory complexes and on transcription repression in vitro. We found that the inducer breaks up the repressosome with clear intermediates in a concentration-dependent manner. The sequential disassembly generates different stages of regulation of the gal operon. The D-galactose-dependent switch from one stage of regulation to another satisfies the amphibolic requirement of the gal operon.2. Regulatory network in galactose amphibolism: Systems biology.The gal regulon of E. coli contains genes involved in galactose transport and metabolism. Transcription of the gal regulon genes is regulated by two iso-regulatory proteins, GalR and GalS, which recognize the same binding sites in the absence of D-galactose. DNA binding by both GalR and GalS is inhibited in the presence of D-galactose. The gal regulon genes are activated in the presence of the cAMP-CRP complex. We studied transcriptional regulation of the gal regulon promoters simultaneously in a purified system to describe the integration of the two signals, cAMP and D-galactose concentration, at each promoter, using Boolean logic. Results show that similarly organized promoters can have different input functions. We also found that at some genes the activity of the promoter and the encoded protein can be described by different logic gates. We combined the transcriptional network of the galactose regulon, obtained from our experiments, with literature data to reconstruct the integrated map of the galactose network. Structural analysis of the network shows that at the interface of the genetic and metabolic network feedback loops are by far the most common motif. (Collaboration with Kim Sneppen, Niels Bohr Institute) 3. Axiom of transcription start point.To investigate the determining factors in the selection of the transcription start points (tsp) by RNA polymerase of Escherichia coli, we systematically deleted or substituted single base pairs (bps) at 25 putative critical positions in the two extended -10 promoters, P1 and P2, of the gal operon. These changes extend downstream from -24 to +1 of the P1 promoter. In vitro transcription assays using supercoiled DNA templates revealed a preference for a purine in the non-template strand for tsp in both promoters. The optimal tsp is the 11th bp counting downstream from the -10 position. A single bp deletion anywhere from -10 to +1 switched the tsp to the next available purine 2-3 bp downstream on the non-template strand whereas deleting a single bp at position from -24 to -11 did not affect the tsp. The nature of the 10 bp sequence of the -10 to -1 region, while affecting promoter strength, did not influence tsp. The cAMP-CRP complex, which stimulates P1 and represses P2, did not affect the tsp selection process. The rules of tsp selection by RNA polymerase containing sigma70 in gal and pyr promoters discussed here may be applicable to others.4. General enhancement of transcription by ribosomal protein S1.Prokaryotic RNA polymerases are capable of efficient, continuous synthesis of RNA in vivo, yet purified polymerase-DNA model systems for RNA synthesis.